Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.

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Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.

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Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research (QB3), San Francisco, CA 94158, USA. Electronic address: stanley.qi@ucsf.edu.

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Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research (QB3), San Francisco, CA 94158, USA. Electronic address: bo.huang@ucsf.edu.

Erratum in

Cell. 2014 Jan 16;156(1-2):373.

Abstract

The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

(A) CRISPR labeling of the non-repetitive region of MUC4 intron 1 using multiple optimized sgRNAs. With 26, 36 or 73 sgRNAs, 1 to 3 spots (arrows) can be detected. (B) Histograms of MUC4 loci counts by CRISPR imaging of the non-repetitive MUC4 sequence. (C) Colabeling of the MUC4 exon 2 and intron 3. The physical proximity (~1 kb) of the two target regions does not increase the puncta number as shown in the histograms. (D) Colabeling of MUC1 and MUC4 genes. Labeling two distal genes (MUC1 on chromosome 1 and MUC4 on chromosome 3 respectively) increases the puncta count as shown in the histograms (n = 20). All scale bars: 5 μm. See also

(A) CRISPR imaging of telomeres in RPE cells (scale bar: 5 μm) and trajectories of three telomeres with different movement modes (scale bars: 200 nm). The trajectory lengths are 600 frames for 1 & 3 and 260 frames for 2. See . (B) Comparison of telomere dynamics using CRISPR (blue) and EGFP-TRF1 (red) labeling in RPE cells. The data are displayed as mean ± standard error. (C) Scatter plot of the CRISPR foci intensity and their microscopic diffusion coefficients. (D) The average MSD curves of telomeres in UMUC3 cells without (blue) and with (orange) hTR. The data are displayed as mean ± standard error. (E) Averaged MSD curves of CRISPR-labeled telomeres in RPE cells measured with scrambled shRNA (blue), TIN2 shRNA (green), or co-expression of TIN2 shRNA and the long (L, red) or short (S, purple) isoform of TIN2. At least 15 cells are analyzed in each case. The data are displayed as mean ± standard error. See also and .

(A) Scheme for analyzing the nuclear localization of the MUC4 gene using both sgMUC4-E3 and sgMUC4-I2(F+E). The nucleus is modeled as an oval and then normalized to a round circle to measure MUC4 positions. (B) The histogram of the normalized MUC4 radial position, r. The nuclear envelope is at the unity position. (C) The histogram of the relative angle of MUC4 loci with respect to the center of the nucleus, θ. 50 cells are analyzed for (B) and (C). (D) Single particle tracking of MUC4 loci movement. See and . (E) Trajectories of the three loci in (D), which show different confinement sizes, Lconfinement, and microscopic diffusion coefficients, Dmicro (scale bars: 200 nm). The trajectory lengths are 900 frames for 2 & 3 and 115 frames for 1. (F) Paired MUC4 loci after DNA replication. See . (G) Histogram of the distance between two MUC4 loci in a pair. 20 cells are analyzed. (H) Long term 3D tracking to measure the pair distances of three MUC4 pairs within a cell. (A), (D), and (F) are 20-frame averages of live recording images. Scale bars: 5 μm. See also and and .

(A) Snapshots from a MUC4 image sequence in which a HeLa cell undergoes mitosis, showing z maximum projections of 4 μm depth. The arrows indicate the MUC4 loci, which are not completely captured during mitosis because the cell thickness exceeds the z range. See . (B) CRISPR labeled HeLa cells fixed and stained with DAPI (blue) to image the relationship between MUC4 loci (green) and the chromosomes. Cells at different stages of mitosis are displayed, showing z maximum projections of 18 μm. Scale bars: 5 μm. See also .